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Date of defense: June 27
thSalmo salar temperature and dietary energy
at the University of Bergen
Dissertation for the degree of philosophiae doctor (PhD)
The material in this publication is protected by copyright law.
Year: 2017
Title: Regulation of appetite and growth of Atlantic salmon (Salmo salar L.) and effect of water oxygen, temperature and dietary energy.
Author: Vibeke Vikeså
Print: AiT Bjerch AS / University of Bergen
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This thesis was carried out at the National Institute of Nutrition and Seafood Research (NIFES) in collaboration with the Institute of Biology at the University of Bergen and Skretting Aquaculture Research Centre (ARC).
The PhD project was financed by two funding programs at Norges Forskningsråd (NFR) with the grant numbers 209655/O30 and 199683/S40 and by Skretting ARC.
Supervisors of the PhD have been Dr Leo Nankervis, Dr Ernst Morten Hevrøy and Prof.
Rune Waagbø.
Acknowledgements
First of all, I thank the Norwegian Research Council (NFR) and my employer Skretting ARC for financing my PhD. I have had great support during my PhD work from all supervisors; Professor Rune Waagbø, Dr Leo Nankervis and Dr Ernst Morten Hevrøy. Your advice, support and input have been valuable and highly appreciated. I would also like to thank my boss, Dr. Wolfgang Koppe who saw this opportunity and encouraged me to start a PhD. All the best for your new business!
The analytical work that I conducted at NIFES would not be possible without the training and support of Eva Mykkeltvedt, Natalia Larsen, Jaap Wessels and Synne Wintertun. Thank you very much for your patience and for taking the time to guide me through the many rounds of real-time and RT-qPCR’s.
I also extend my thanks to the helpful colleagues at Skretting ARC laboratory and Lerang Research station, who always tried to seek the best solutions to any issues relating to fish, samples and experimental quality.
My colleague, Dr. Saravanan Subramanian has been of invaluable help in writing of papers and my thesis. Thank you so much for your quick responses and constructive input on manuscripts. I would also like to thank my colleagues, Sophie Noonan and Nafiha Usman, for their superb help with illustrations and statistical support.
Support from family, friends and good colleagues has been essential for me to complete this PhD. To keep my spirits up while finalising the thesis, my colleague Dr Guido Riesen has treated me with Friday croissants for several months. Thanks, it was highly appreciated!
I would especially like to thank my good friends, Linda and Ellen for the support and great believe in me. I would also like to extend my appreciation to Ellen and her family for always welcoming me to stay at their house during the many visits to Bergen.
And last, but not least, a big thank you to my boyfriend Kjell Erik and son Bror, for happy times and lots of fun!
Abstract
High water temperature combined with low dissolved oxygen (LO or hypoxia) is one of the most challenging environmental conditions farmed fish experience. The oxygen requirement of fish increases in parallel to this, which limits the aerobic energy metabolism and consequently reduces feed intake and growth of fish. The global ocean warming followed by reduced oxygen availability, is expected to exacerbate associated physiological stress on fish in several areas where Atlantic salmon are currently farmed. Understanding the impact of temperature and limited oxygen on growth regulatory mechanisms and the energy metabolism, will be of significant relevance to both cultured and wild fish populations.
Conditions of high temperature and hypoxia are related with reduced feed intake and growth in fish. It is unclear whether the low oxygen availability directly affects growth regulatory mechanisms, and if low feed intake is the primary cause of depressed growth under LO conditions. Studies of appetite and growth regulation in salmon under such conditions are few, and considerations of the fluctuating character of endocrine signals and nutrient absorption are scarce. Limitation of the aerobic energy metabolism under reduced oxygen availability is further restricted by a thermal increase. It is therefore interesting to find out how high energy diets can potentially impact appetite and growth regulation under LO conditions.
This thesis therefore investigated mechanisms by which LO and high temperature conditions impact appetite and growth regulation in seawater adapted Atlantic salmon. Free amino acid (FAA) and endocrine dynamics in relation to meal time were also studied. Four fish trials were conducted, including the following variables; dissolved oxygen (DO; LO and high, HO), temperature and digestible energy (low and high, LE and HE). Endocrine appetite and growth signalling was investigated through analyses of plasma ghrelin and IGF-1 concentration, and mRNA levels of the growth hormone receptor (ghr1) and insulin like growth factor-1 (igf1) in liver and muscle tissue.
LO conditions demonstrated direct depressed effects on appetite and growth in salmon across temperatures. Reduced growth in salmon under LO was not caused only by a reduced
feed intake, but appeared to be a combined effect of impairment of growth regulation and increased metabolic costs, as demonstrated by a pair-feeding technique. Increased metabolic costs by LO were indicated by responses in oxyregulating mechanisms, such as increased haemoglobin, reduced blood pH and imbalanced osmoregulation. Reduced specific growth rate (SGR) and feed intake were also found for salmon under LO compared to HO groups, at both optimal and high temperatures. High temperature demonstrated a diminished growth potential in salmon compared to an optimal temperature. This was reflected in a faster 24 hour postprandial catabolism of absorbed FAA, and generally lower and faster declines of IGF-1 (plasma and mRNA) at 19°C compared to 13°C.
Ghrelin was found to signal feed anticipation in salmon, consistent with mammalian findings, and reflected by clear preprandial plasma ghrelin peaks at 12°C. Ghrelin and GH- IGF factors responded to LO at a high temperature, but further studies should focus on a postprandial perspective to confirm the preprandial peaks at 12°C.
Results from feeding HE diets to salmon, indicate that it is possible to stimulate growth, feed utilisation and the energy metabolism under LO conditions through DE level, regardless of temperature.
To summarise, the thesis shows that growth regulation in seawater adapted salmon is negatively affected by LO at optimal and high temperatures. Positive effects from feeding HE diets under LO, demonstrate possibilities to support energy metabolism through dietary means under challenging environmental conditions. Diet effects and environmental impact on growth regulation are of great relevance to salmon farming, as further knowledge can improve growth, welfare, health and future farming possibilities.
List of publications
Paper I
Vikeså V., Nankervis L., Remø S.C., Waagbø, R. & Hevrøy, E.M. (2015). Pre and postprandial regulation of ghrelin, amino acids and IGF-1 in Atlantic salmon (Salmo salar L.) at optimal and elevated seawater temperatures. Aquaculture 438, 159-169.
Paper II
Vikeså, V., Nankervis, L. & Hevrøy, E.M. (2017). High dietary energy level stimulates growth hormone receptor and feed utilization in large Atlantic salmon (Salmo salar L.) under hypoxic conditions. Aquaculture Nutrition In press.
Paper III
Vikeså, V., Nankervis, L. & Hevrøy, E.M. (2016). Appetite, metabolism and growth regulation in Atlantic salmon (Salmo salar L.) exposed to hypoxia at elevated seawater temperature. Aquaculture Research 12, 1-16.
Reprints of the three papers were made with permission from the publishers; Elsevier (Paper I) and Wiley (Paper II and III).
Contents
SCIENTIFIC ENVIRONMENT ... 2
ACKNOWLEDGEMENTS ... 3
ABSTRACT ... 4
LIST OF PUBLICATIONS ... 6
CONTENTS ... 7
1. INTRODUCTION ... 8
1.1 GENERAL INTRODUCTION ... 8
1.2 THERMAL TOLERANCE AND AEROBIC SCOPE ... 9
1.3 THERMAL TOLERANCE ... 9
1.4 OXYGEN ... 14
1.5 INFLUENCE OF NUTRITIONAL FACTORS UNDER WARM WATER CONDITIONS ... 21
1.6 ENDOCRINE REGULATION OF APPETITE AND GROWTH ... 23
1.7 APPETITE REGULATION BY GHRELIN ... 25
1.8 GROWTH REGULATION BY THE GH-IGF SYSTEM ... 29
2. AIMS OF THE THESIS ... 36
3. ABSTRACT OF PAPERS ... 37
3.1 PAPER I ... 37
3.2 PAPER II ... 38
3.3 PAPER III ... 38
4. GENERAL DISCUSSION ... 40
4.1 HIGH TEMPERATURE ... 42
4.2 APPETITE REGULATION ... 48
4.3 LOW OXYGEN AVAILABILITY ... 54
4.4 CAN DIETARY FACTORS IMPACT PERFORMANCE UNDER HYPOXIA AND HIGH TEMPERATURE? 63 5. CONCLUSIONS ... 68
6. FUTURE PERSPECTIVES ... 70
REFERENCES………...………...………..……… 71
PAPERS……….……….. 86
1. Introduction 1.1 General introduction
Globally Atlantic salmon production exceeds 2.3 million tonnes per annum (FAO, 2014a).
However, its farming is restricted to geographical areas with a suitable environment.
Favourable farming conditions are characterised by deep waters with a high water exchange and invariable weather conditions, providing stable cool and oxygen-rich seawater, within optimal ranges for salmon growth. Since its beginning in Norway in the 1960’s, salmon farming has grown to a great extent in both northern and southern hemispheres including regions in Scotland, Ireland, the Faroe Islands, North America, Chile and Tasmania (Australia) (FAO, 2014a). The world population increasingly relies on farmed fish to meet their requirement for long chain polyunsaturated (n-3) fatty acids and proteins, and the share of farmed fish in global fish consumption by humans is projected to exceed 53% by 2022 (FAO, 2014b). Forecasted global climate change revolves heavily around temperature change (Perry et al., 2005; Pörtner and Knust, 2007) and predicts negative impacts on wild fisheries, land-based food production (Ficke et al., 2007; IPCC, 2014) and coastal water quality, including increased prevalence of hypoxic conditions (Diaz, 2001; Diaz and Breitburg, 2009; Rabalais et al., 2009). This underlines the importance of knowledge generation relating to ensuring food production under suboptimal environmental conditions to facilitate a continued supply of farmed fish.
Global ocean oxygen content has decreased significantly due to warmer sea temperatures following the 1950’s (Helm et al., 2011; IPCC, 2014), while increased river temperatures (Daufresne et al., 2009; Durance and Ormerod, 2007; 2009; Elliott and Elliott, 2010) indicate the same trend in freshwater. Climate models predict an increase in average surface temperatures of 1.0 to 5.0°C in Southern Hemisphere seawater and 1.5 to 11.0°C in Northern Hemisphere seawaters by 2100 (IPCC, 2014). This is expected to exacerbate associated physiological stress on fish in several areas where Atlantic salmon are currently farmed. In fact, the largest salmon farming countries, Norway and Chile, are considered the
top two marine production areas with highest vulnerability to associated climate changes in the world (Handisyde et al., 2016). Understanding the implications of climate change on growth regulatory mechanisms will have an increasing relevance to both cultured and wild fish populations globally.
1.2 Thermal tolerance and aerobic scope
Water oxygen and temperature are the major environmental factors affecting metabolic rate and growth potential of fish (Brett, 1979). While temperature directly controls metabolic rate, oxygen availability in water has a limiting role on energy metabolism, below a certain threshold level (Brett, 1979). The solubility of oxygen is reduced when temperature increases, and so warm conditions, such as summer and late autumn, is commonly accompanied with periods of low water oxygen.
1.3 Thermal tolerance
Thermal tolerance window is defined as a species specific temperature range in which the animal thrives and grows, and with additional upper and lower temperature ranges defined as lethal or critical. The width of the thermal window generally varies with size or life stage, starting with a narrow window which widens at later stages and then a narrowing, i.e. a reduced thermal tolerance, for larger specimens and during reproduction (Pörtner et al., 2006; Pörtner and Farrell, 2008) (Fig 1.1).
Conversion efficiency and rates of ingestion, growth and metabolism are accelerated by increasing temperature, peaking at different optimal temperatures before declining and eventually ceasing if peaking at the upper thermal limit (Brett, 1979; Jobling, 1997) (Fig 1.2).
Figure 1.1. The aerobic thermal window across life stages for fish. Modified from Pörtner and Farrell (2008).
Figure 1.2. Effect of temperature on feed conversion efficiency (A) and rates of growth (B), ingestion (C) and metabolism. The letters (A, B, C) indicate respective maxima (optimum, peak of the curves) in relation to temperature.
Modified from Jobling (1997).
Recommendations on how to optimise feeding and growth under different temperature and oxygen levels are lacking and would benefit both welfare and growth conditions of farmed fish. In this context, it is especially effects from fluctuating environmental conditions that should be investigated, as this is more representing the conditions fish are facing in the open sea.
Fish body temperatures rely on the surrounding water temperature, to which they are sensitive enough to detect changes as small as 0.03°C (Murray, 1971). Sensitivity to temperature changes can be an advantage, especially for wild fish, as they can respond by swimming away from waters of a suboptimal quality. Farmed salmon are more confined to the limit of their sea cages, and have restricted scope for migration to more favorable environments.
Large fish are generally considered more vulnerable to thermal stress than smaller fish (Fig 1.1), but this is debated (Clark et al., 2012; Clark et al., 2008; Daufresne et al., 2009; Elliott, 1981; Lefevre, 2016; Nilsson and Östlund-Nilsson, 2008; Pörtner and Knust, 2007).
Under short-term temperature fluctuations, large fish are claimed to have an advantage due to a smaller skin surface area in relation to body volume (Elliott, 1981). Internal temperature is exchanged mainly via the skin, and to a lesser degree through the gills (Elliott, 1981), and so a large body size requires longer time to adjust body temperature to ambient water temperature than smaller fish. This will then result in reduced effects on body temperature and metabolism compared to smaller fish (Elliott, 1981).
However, there is a faster reduction in growth and survival of large fish than small fish when thermal tolerance is limited by hypoxia (Pörtner and Knust, 2007). Large fish are therefore less robust when facing long term environmental changes and handling stress than smaller fish. This is supported by lower abundance and survival of large fish in wild fish populations exposed to prolonged water temperature increase (Daufresne et al., 2009;
Pörtner and Knust, 2007). In fact, a reduced fish size is recognised as a common
consequence of global warming to aquatic animals, in addition to shifts in habitat and disorder in seasonal life cycle sequences (Daufresne et al., 2009).
Farming of salmon stands the biggest economic risk when large salmon, approaching the end of the production cycle, are exposed to suboptimally warm and hypoxic conditions.
Future research on metabolic regulation of this size group is therefore of great interest.
Acclimation to a wide temperature range can increase tolerance to critical temperatures, but has little effect on preferred or optimal temperature limits (Pörtner and Peck, 2010). This ability to adapt tolerance to thermal extremes can be an advantage at locations of frequent temperature changes and towards global warming.
Salmon farms experience changes in temperature due to stratification and oscillations occurring naturally across seasons in periods lasting from days to months. In parallel, salmon in cages are also exposed to significant variations in oxygen and light (Johansson et al., 2006). Within the water column of a cage, the temperature is generally higher closer to the surface during the warm season and the opposite in the cold season (Oppedal et al., 2007). Episodes of ice melting or heavy rain can alter this and create the opposite situation in fjords with high run off from land. Daily mean temperature in the fjords during a Norwegian summer or autumn can vary from 7-15°C (Aas-Hansen, 2010) with maximum reaching up to 20°C (Johansson et al., 2006; Oppedal et al., 2011), while Tasmanian salmon farming (Australia) holds mean summer temperatures of 17-21°C (Attard et al., 2012; Carter et al., 2008).
Temperatures optimal for growth are size and life stage and species dependent (Jobling, 1981). Literature on optimal temperatures varies, but the principles of the thermal window (Pörtner et al., 2006; Pörtner and Farrell, 2008) (Fig 1.1) generally applies to Atlantic salmon. Eggs have the narrowest thermal window (4-7°C and critical temperature at 7-8°C) (Elliott and Elliott, 2010), while a range of 16-20°C is reported optimal for the freshwater stages (typically 0-100 g), compared to 12.8-14°C for the postsmolt stage (50-300 g) (Coutant, 1977; Elliott and Elliott, 2010; Elliott and Hurley, 1997; Forseth et al., 2001;
Handeland et al., 2008; Koskela et al., 1997; Peterson and Martin-Robichaud, 1989).
Optimal growth of large Atlantic salmon (1.6-2.9 kg) reared in tanks were found at 13°C compared to 15°C, 17°C and 19°C (Hevrøy et al., 2013).
Temperature effect on growth is highly dependent on feed intake (Jobling, 1997). A compromised feed intake (i.e. less ingested energy) can lower the fish’s thermal tolerance (Elliott and Elliott, 2010; Jobling, 1997) and reduce the optimal temperature for growth (Elliott and Hurley, 2000a; b). A limitation in feed ration or a reduced feed intake also lower growth rate and so feed access must be unlimited to allow maximum growth. Elevated water temperature also reduces feed intake when temperature rises above preferred thermal optimum values (Brett, 1979). While optimal temperature for feed conversion (a) is slightly lower than that for maximum growth (b), the highest feed ingestion rate (c) is achieved at a little higher temperature than that for maximum growth (b) (Brett, 1979; Jobling, 1997) (Fig 1.2).
1.4 Oxygen
The impact of warm water on fish physiology cannot be evaluated without considering the water oxygen concentration, since the physicochemical characteristics of these parameters in water are closely connected. Oxygen solubility in water (DO) is reduced at higher temperatures (Brett, 1979), so raising temperature naturally lowers DO concentration (mg L-
1), even if it is maintained at 100 % oxygen saturation (Nilsson, 2010) (Fig 1.3). Oxygen is a limiting factor for fish metabolism due to its crucial role in aerobic synthesis of ATP and consequently regulates energy availability for metabolism.
Figure 1.3. Dissolved oxygen concentration (DO; mg L-1) at 100 % oxygen solubility (saturation) in freshwater and seawater in relation to temperature. Modified from source: Nilsson (2010).
Low dissolved water oxygen, also called environmental hypoxia, is recognised by a shortage of oxygen below the requirement for physiological functions of an organism (Farrell and Richards, 2009). Hypoxic water, normally followed by the build-up of excreted CO2 and NH3, impacts growth in fish at critical levels (Thorarensen and Farrell, 2011).
Saturation level and duration of exposure decide when DO becomes critical for the fish (Nilsson and Randall, 2010). At the extremes, when oxygen levels are low (hypoxic) or absent (anoxic), fish metabolism goes from an anaerobic to anoxic state (Claireaux et al., 2000), and eventually death (Pörtner and Farrell, 2008; Pörtner and Knust, 2007).
An elevation in water temperature also increases oxygen consumption by fish (Barnes et al., 2011). The challenge for fish under warm conditions is therefore to meet an increasing oxygen demand under decreasing DO. Fish living in the marine environment are even more susceptible to low oxygen due to a lower DO in sea compared to freshwater (Fig 1.3).
As for thermal stress, also oxygen sensitivity according to fish size is debated. Large fish are considered to cope better than smaller fish under severe episodes of hypoxic and anaerobic conditions (Nilsson and Östlund-Nilsson, 2008). This is due to a lower metabolic rate and decreasing oxygen demand with increasing body mass, which means that easily accessible energy stores (glycogen) last longer and accumulation of unfavourable levels of lactic acid is slower than in smaller fish (Nilsson and Östlund-Nilsson, 2008). On the contrary, large body size is also claimed to be a disadvantage under hypoxic conditions due to a faster depletion of dissolved oxygen than in smaller fish (Pörtner and Peck, 2010).
Blood oxygen levels are generally lower in larger fish (Clark et al., 2012; Clark et al., 2008), which require longer restoration time following hypoxia and stress handling (Clark et al., 2012).
However, in a review of body size and hypoxia tolerance, it was concluded that size did not matter in terms of oxygen uptake, since reduced gill surface in larger specimens is also followed by a reduced metabolic rate (Nilsson and Östlund-Nilsson, 2008) and a reduced oxygen consumption relative to body size (Gjedrem, 1993).
The aerobic scope (AS) is the capacity to consume oxygen from a calm state (standard metabolic rate, SMR) to a maximum (MMR), which indicates how much oxygen is available for swimming, behaviour, immune functions, reproduction and growth processes (Farrell and Richards, 2009; Pörtner and Peck, 2010; Rogers et al., 2016) (Fig 1.4). Optimal growth conditions are met at maximum AS within the species thermal window. As a consequence of decreasing oxygen availability and a reduced AS, the activity of vital functions are reduced or ceased, but how exactly this down regulation progress in fish is not known (Farrell and Richards, 2009). Water oxygen supply becomes critical (Pcrit) when the
oxygen uptake cannot meet the requirement of SMR, which only supports very basic activities, and the metabolism shifts to anaerobic energy dependency (Pörtner, 2010).
Figure 1.4: Diagram illustrating aerobic scope (AS) and metabolic responses of fish to dissolved oxygen (DO). Solid lines indicate O2 consumption rates to support standard (SMR), routine (RMR), maximal metabolic rate (MMR) and AS of an oxyregulating fish. AS is the difference between MMR and SMR, which decreases with increasing hypoxia.
Dashed lines show theoretical effects of decreases in DO level on physiological functions. Below critical dissolved oxygen (DOcrit) of RMR and SMR the metabolism becomes dependent on water O2 and anaerobic energy. DOcmax is defined as the critical DO level at which oxygen supply no longer meets the maximum demand for oxygen. Modified from Farrell and Richards (2009) and Pörtner (2010).
Recent studies on AS at a high temperature indicate that acclimation can prevent an acceleration of metabolic rate and maintain the AS in fish species (Gräns et al., 2014; Norin et al., 2014; Raby et al., 2016; Sandblom et al., 2014). A long term elevation in temperature may therefore maintain the growth potential better than under a sudden period of warming.
It also highlights awareness of thermal adaptation time when interpreting growth results from high temperature fish studies.
The concept of oxygen and capacity-limited thermal tolerance (OCLT) describes how the aerobic scope and the thermal window interrelates (Pörtner and Peck, 2010) (Fig 1.5).
Oxygen requirement is met within the thermal window, but as temperature increases the oxygen consumption also goes up (Barnes et al., 2011). When approaching the extremes of the thermal window, i.e. critically low or high temperature (Tcrit), the aerobic scope limits
the performance and puts constraints on the metabolism due to reduced oxygen supply (Fig 1.5) from (Pörtner and Farrell, 2008; Pörtner and Knust, 2007). A raise in temperature increases Pcrit, i.e. oxygen supply becomes limited at a higher level than Pcrit at a lower temperature, which is demonstrated in Atlantic salmon (Barnes et al., 2011). Increasing hypoxia and carbon dioxide levels reduce both thermal window width and aerobic scope (Pörtner and Peck, 2010).
Figure 1.5. Model of how aerobic scope (AS) and metabolic rate are limited by thermal changes, i.e. the concept of oxygen and capacity-limited thermal tolerance (OCLT). AS is the difference between maximum metabolic rate (MMR) and standard metabolic rate (SMR), which is limited by critical low and high temperatures (Tcrit Low and Tcrit High) and oxygen supply. AS is at maximum (Max) at an optimal temperature (grey dashed line). Source: Pörtner and Peck (2010).
The most predictable occurrence of hypoxia in commercial salmon farming is under conditions of high water temperature, but low and fluctuating oxygen also occurs in connection to shallow water, low tidal current, stratification, algae bloom, short day length, high fish biomass density and during feeding throughout the year.
Growth and feed utilisation of seawater adapted salmon improved when oxygen was raised from 50% to 100% saturation at 8-9°C (Bergheim et al., 2002) and reflects the importance of available oxygen for performance of this species. Growth of salmon is significantly
improved by raising oxygen saturation from 70-75% to 80-85% at 15°C (Bergheim et al., 2006) and oxygen level below 70% saturation reduces feed intake at 16°C (Remen et al., 2012). A review by Thorarensen and Farrell (2011) mentions that although recommended oxygen level for optimal growth of salmonids is minimum 70-80% saturation, it is close to 100% saturation to gain maximum growth of salmon. Salmon growth is found to increase even further at oxygen levels up to approximately 120 % (Hosfeld et al., 2008). A recent study of salmon postsmolt (Remen et al., 2016) demonstrates well how level of oxygen required to achieve maximum feed intake is strongly dependent on temperature. At a low temperature (7°C) maximum feed intake was found at 42 % DO, 11°C at 53% DO, 15°C at 66% and at 76% DO for the highest temperature (19°C) (Remen et al., 2016). These results are consistent with Atlantic salmon being increasingly sensitive to low oxygen water at high temperatures.
Fish can adapt to hypoxia by behavioural and physiological adaptations depending on species, size, habitat and degree of the hypoxia (Chapman and McKenzie, 2009). The behavioural responses to environmental changes by farmed fish are spatially limited in the sea cages. Positioning of fish in the water column is affected by temperature, oxygen and light, among other factors (Johansson et al., 2006; Oppedal et al., 2011; Oppedal et al., 2007; Oppedal et al., 2001). Fish energy metabolism has two coping mechanisms for declining oxygen availability depending on degree and duration; oxyregulation when oxygen is approaching, but is still above Pcrit, and oxyconforming under anaerobic conditions below Pcrit (Pörtner, 2010).
Oxyregulation is defined by behavioural, physiological or anatomical adaptations to maintain a steady oxygen supply to meet the demands of aerobic energy production without making any compromises in metabolic rate (Farrell and Richards, 2009; Perry et al., 2009).
The most prominent physiological adaptation is the hypoxic ventilator response which is an immediate gill ventilation initiated by oxygen sensing cells (neuroepithelial) in gill filaments (Perry et al., 2009). Under long-term hypoxia, these cells can increase in density and change morphologically to facilitate oxygen uptake (Nilsson, 2007; Perry et al., 2009).
Progression in hypoxia stimulates accompanying mechanisms supporting uptake and
transport of oxygen which can also relieve the degree of hyperventilation (Perry et al., 2009).
An increase of haemoglobin (Hb) concentration is the most common mechanism for fish to improve uptake and carrying capacity of oxygen and increase tolerance to hypoxia (Nilsson, 2007; Nilsson and Randall, 2010; Perry et al., 2009; Stevens et al., 1998). Additionally, fish can also increase mean red blood cell volume (MCV), red blood cell count (RBC) or haematocrit concentration (Hct) to support oxygen supply under low oxygen conditions (Perry and Gilmour, 2010). Hb’s affinity for oxygen is reduced with higher temperatures, and so the ability to regulate its affinity to oxygen can be aiding delivery of oxygen to the tissues. Anaerobic metabolism reduces blood pH (Nilsson and Randall, 2010), which reduces oxygen affinity and consequently releases more oxygen to the tissue (Jensen et al., 1998). Changes in haematology and osmoregulation are known as secondary responses to environmental stress (Barton, 2002). Imbalances in ions may also impact the oxygen binding capacity of Hb, as an increased flow of Na+ into red blood cells can inhibit binding of oxygen to Hb, releasing more oxygen to the metabolism (Ferguson and Boutilier, 1989;
Ferguson et al., 1989).
Oxyconforming is a reduction of metabolic rate so that oxygen is used more sparingly (Nilsson and Randall, 2010; Perry et al., 2009; Ultsch et al., 1981). Reduced feed intake is perceived as a consequence of oxyconforming and is thought to be a major cause of depressed growth of fish under low DO conditions (Brett, 1979; Carter et al., 2008), while secondary mechanisms affecting growth under low DO conditions are yet to be well elucidated.
Atlantic cod (Gadus morhua) exposed to hypoxic conditions during feeding, immediately stopped eating when the declining oxygen uptake approached the critical oxygen level (Pcrit, Fig 1.4), but resumed feeding activity when oxygen saturation reached normoxia again (Claireaux et al., 2000). Claireaux et al. (2000) suggest that this demonstrates that the behavioural adaption of reducing feed intake can support metabolism in maintaining its aerobic scope under increasingly energy demanding conditions. Another study with cod (Chabot and Dutil, 1999) showed that reduced feed intake was responsible for 97% of the
growth reduction going from a normoxic to a hypoxic condition. Reducing feed intake is no doubt an efficient energy saving mechanism since digestion of food in cod can require up to 90% of its aerobic scope (Soofiani and Hawkins, 1982).
Atlantic salmon are considered to have a poor tolerance to low oxygen, based on their naturally active life style in oxygen rich habitats, with limited capacity to oxyregulate under severe hypoxia. However, postsmolt demonstrated a high ability to oxyregulate under hypoxia at 14°C, while use of oxyconforming mechanisms dominated under hypoxia at higher temperatures (18-22°C) (Barnes et al., 2011). This indicates that there are still more to be elucidated in terms of hypoxia tolerance in Atlantic salmon.
1.5 Influence of nutritional factors under warm water conditions
Feed consists of mainly protein, fat, carbohydrates and moisture, but before the body can make use of ingested feed the nutrients are broken down to smaller units in the gut to enable absorption into the blood. In this way, the metabolism is provided with essential nutrients, where absorbed nutrients provide easy accessible energy for metabolism. Absorbed amino acids are transported via the circulatory system into the white muscle pool. Free amino acids in white muscle tissue indicate substrate availability for muscle growth (Espe et al., 1993).
Post-digestive absorption of amino acids is also thought to stimulate endocrine growth promoting processes and signalling satiety (Mommsen, 2001; Planas et al., 2000).
Several studies report on postprandial concentrations of free amino acids (FAA) in plasma and tissues of Atlantic cod (Gadus morhua) (Lyndon et al., 1993), koi carp (Cyprinus carpio) (Kwasek et al., 2010; Ogata, 1986), rainbow trout (Salmo gairdnerii R./
Oncorhynchus mykiss) (Barrows et al., 2007; Carter et al., 1995; Kaushik and Luquet, 1979;
Larsen et al., 2012; Schlisio and Nicolai, 1978; Yamada et al., 1981; Yamamoto et al., 2005) and Atlantic salmon (Carter et al., 2000; Espe et al., 1993; Mente et al., 2003; Sunde et al., 2003). However, data are lacking on amino acid flux in Atlantic salmon at elevated environmental temperatures.
Fat is the most energy-dense macronutrient and the energy yield is more than double that of proteins or carbohydrates (Schmidt-Nielsen, 1997a). Dietary digestible energy is therefore readily optimised by changing lipid inclusion and choice of raw materials. Dietary fat require less oxygen for growth (fat deposition) than protein or starch, which is shown for both Nile tilapia (Oreochromis niloticus) and rainbow trout (Oncorhynchus mykiss) (Saravanan et al., 2013a; Saravanan et al., 2012). This will result in a higher DE intake compared to DE supplied mainly as protein or starch in the diets (Saravanan et al., 2013a;
Saravanan et al., 2012).
Dietary protein and fat recommendations are based on studies under normoxic conditions.
However, the protein growth potential of salmon is reduced under limited oxygen availability. Feeding salmon with high protein under hypoxia, will then require fish to
catabolise access protein above requirement, and convert it into fat. This will demand more oxygen than utilising a diet that meets the requirement. Therefore, it is possible that a reduction in dietary protein and increase in fat, might support the oxygen budget under hypoxia, since fat can be deposited with less oxygen use than protein and starch. This could be an interesting diet to test under low oxygen conditions, especially for large salmon, due to the higher fat deposition than in smaller fish (Shearer et al., 1994). An additional thermal elevation puts an even further demanding challenge on salmon. It is therefore of particular interest to determine how dietary energy interacts with oxygen availability to regulate feed intake and growth under such conditions.
1.6 Endocrine regulation of appetite and growth
The endocrine system activates and maintains balance of important physiological functions like digestion, metabolic reactions, osmoregulation, appetite, growth, reproduction and behaviour.
Fish generally have the same endocrine organs as mammals and other vertebrates, except for the corpuscles of Stannius and urophysis, which are specific for fish species (Bone and Moore, 2008) (Fig 1.6).
Figure 1.6. The endocrine organs of teleost fish. Modified from: Bone and Moore (2008).
Hormones, which are the chemical messengers of the endocrine system, are released from specialised glands in the brain (neuropeptides) or other organs into the blood stream. They are then transported to a peripheral target tissue (endocrine), a neighbouring cell (paracrine) or inside the cell it is secreted from (autocrine). Hormones are recognised by specific receptors on the target which signals the hormone to carry out its function or stimulate the release of other hormones (Schmidt-Nielsen, 1997b). Several target organs can be affected by a single hormone and also one target organ can be affected by several hormones. This makes it useful to study the interactions of hormones related to the response of interest.
The endocrine messengers can be categorised as fat-soluble (steroids) and water-soluble (peptides, proteins and tyrosine-derived hormones) based on chemical composition and characteristics. Endocrine regulation operates by interacting with the central nervous system, called neuroendocrine regulation.
The pituitary has a central role in hormonal regulation of diverse physiological functions, including growth, by controlling the secretion of selected hormones as well as producing its own. The gastrointestinal tract (GIT) is the largest endocrine organ and interacts with the brain (the brain-gut axis) to govern a number of endocrine reactions dealing with physiological functions like appetite, digestion, absorption and osmoregulation. One of the most profound neural influences on the endocrine signalling pathway is the hypothalamus’
regulation of the pituitary gland (Bone and Moore, 2008). The hypothalamus is also the principle regulator of appetite in vertebrates including fish species (Kulczykowska and Sánchez Vázquez, 2010).
Endocrine function is vulnerable to stressors such as changes in environmental parameters (Pickering et al., 1991) making them relevant response indicators as well as functional descriptors of physiological processes. Sensitivity of fish to stressors also underlines the importance of having optimal adaptation conditions prior to sampling to rule out confounding factors.
1.7 Appetite regulation by ghrelin
Appetite is regulated by stimulating (orexigenic) and inhibiting (anorexigenic) hormones (Fig 1.7). Known orexigenic hormones in fish are apelin, galanin, ghrelin, growth hormone (GH1), neuropeptide Y (NPY) and orexins (Volkoff, 2016; Volkoff et al., 2010). The most studied anorexigenic factors include leptin, cocaine- and amphetamine-regulated transcript (CART), melanin-concentrating hormone (MCH) and cholecystokinin (CCK) (Volkoff et al., 2010).
While ghrelin studies within the Cyprinidae family are many and conclusive, the role of ghrelin in feeding regulation in salmonids is still not defined, according to a recent review by Volkoff (2016) (Fig 1.7).
Figure 1.7. Key stimulating (orexigenic) and suppressing (anorexigenic) regulators of feeding in the most studied fish family Cyprinidae, and Salmonidae. Middle column lists factors where no effect on feeding is established. The symbol
? indicates uncertain role in regulation of feeding. Modified from Volkoff (2016) and Volkoff et al. (2010).
Ghrelin was first identified as a strong growth stimulant, hence the name, which is derived from the Proto-Indo-European word “ghre” meaning growth. Ghrelin was first isolated from rat stomach tissue, where it was identified as the natural ligand that specifically binds to the growth hormone secretagogue receptor (GHSR) in the pituitary (Kojima et al., 1999). This is also called the ghrelin receptor, which stimulates the secretion of pituitary GH1 (Fig 1.8).
In the same study, human ghrelin was found to resemble rat ghrelin except for two amino acids (Kojima et al., 1999). This led to a cascade of identifications of ghrelin and its GH1 stimulating function in a wide range of vertebrates (Kojima and Kangawa, 2005), mammalian and non-mammalian, including a great number of fish species like goldfish (Unniappan et al., 2002), eel (Kaiya et al., 2003c), tilapia (Kaiya et al., 2003b), rainbow trout (Kaiya et al., 2003a), channel catfish (Kaiya et al., 2005), black sea bream (Yeung et al., 2006), cod (Xu and Volkoff, 2009) and Atlantic salmon (Hevrøy et al., 2011; Murashita et al., 2009).
Fish ghrelin varies in peptide size from 12-26 amino acids, while human ghrelin has 28 amino acids. Fish species can also have more than one molecular form of ghrelin including variations in the peptide structure (Kojima et al., 2008; Murashita et al., 2009). Activation of the biological function of ghrelin relies on a modification of the 3rd amino acid; usually serine, by acylation with a medium chained fatty acid (typically n-octanoic acid or n- decanoic) (Kojima et al., 2008).
As the only orexigenic hormone, ghrelin is synthesised in the gastrointestinal tract (GI), primarily in the stomach, but also in the brain (hypothalamus and pituitary), pancreas and heart tissue (Fig 1.8). Expression of ghrelin in teleost fish are also found in the spleen, liver, kidney and gills (Kaiya et al., 2008). In Atlantic salmon, expression of ghrelin has also been detected in adipose tissue, but its exact role is not yet described in this context (Murashita et al., 2009).
The role of ghrelin as a powerful appetite stimulant started out with the findings of elevated plasma ghrelin levels during fasting and a postprandial decline in humans (Cummings et al., 2001; Inui, 2001; Tschöp et al., 2001). Since then ghrelin has also received a lot of attention
in fish studies as the main hormone responsible for appetite stimulation (Frøiland et al., 2010; Hevrøy et al., 2012; Jönsson, 2013; Matsuda et al., 2006a; Matsuda et al., 2006b;
Miura et al., 2006; 2007; Riley et al., 2005; Unniappan and Peter, 2005). A continuously growing number of ghrelin studies, show that this is a multifunctional peptide also in fish (Kaiya et al., 2008). Ghrelin interacts with other hormones, and stimulates synthesis of GH1, prolactin (PRL), somatolactin (SL), follicle stimulating hormone (FSH) and luteinising hormone (LH) (Kaiya et al., 2008). It is also involved in swimming and feeding behaviour and several physiological functions (Kaiya et al., 2008).
Ghrelin also holds a well-known role in energy balance in fish and higher vertebrates (Choi et al., 2003; Cummings, 2006; Cummings and Shannon, 2003; Frøiland et al., 2010; Hevrøy et al., 2012; Inui et al., 2004; Jönsson, 2013; Kaiya et al., 2008; Riley et al., 2005; Ueno et al., 2005; Unniappan and Peter, 2005). Correlations to body fat and increased feed intake and body weight following ghrelin treatment suggested an involvement of ghrelin in energy balance (Cummings, 2006; Tschöp et al., 2000).
Leptin is also involved in energy metabolism and in mammals has a counteracting function to ghrelin by imposing satiety (Zigman and Elmquist, 2003). Leptin in mammals is secreted from adipose tissue and signals body fat status to the brain via circulation to induce anorexia (Saladin et al., 1995; Zigman and Elmquist, 2003). In contrast, fish leptin seems to be of mainly hepatic origin (Huising et al., 2006; Kurokawa et al., 2005) and its functions in energy metabolism are not yet fully described (Rønnestad et al., 2010). Studies of leptin show a link to anorexia and responses to feed availability in various fish species (Kling et al., 2009; Moen and Finn, 2013; Murashita et al., 2011; Rønnestad et al., 2010; Won et al., 2013). This implies that leptin has a role in satiety signalling in fish as well as in mammals.
Appetite signalling to encourage fish to eat is mediated by circulating ghrelin signalling the brain via the central nervous system, by interacting or stimulating release of other appetite regulating peptides from the hypothalamus (Fig 1.8) (Hosoda et al., 2006; Kulczykowska and Sánchez Vázquez, 2010), or by working directly on tissue nearby its production site.
Appetite and growth are integrally linked, with appetite providing stimulus to eat and
thereby the nutrients required for growth. This is exemplified by the central role of ghrelin in stimulating both appetite and GH1 release (Inui et al., 2004; Kling et al., 2012). Ghrelin is therefore a suitable candidate to study interactions between appetite regulation and the GH-IGF system (Kaiya et al., 2009). Concentration of ghrelin in plasma was analysed in this thesis (Fig 1.8).
Behaviour of swimming and feeding, and hormones and genes related to feeding and energy metabolism (ghrelin, GH secretagogue receptor, leptin, orexin, neuropeptide Y, melatonin) are shown to interact with the circadian system in many fish species (Betancor et al., 2014;
Boujard and Leatherland, 1992; Feliciano et al., 2011; Hoskins, 2011; Kulczykowska and Sánchez Vázquez, 2010; Nisembaum et al., 2012; Rensing and Ruoff, 2002; Vera et al., 2013). This means that several functions display a daily and seasonal rhythm, and progression of hormones and their receptors over time can add valuable information to endocrine studies.
1.8 Growth regulation by the GH-IGF system
Fish growth regulation involves a range of hormones, with those receiving the most attention belonging to the GH-IGF system; specifically growth hormone (GH1 also called somatotropin) and insulin like growth factor-I (IGF-1) (Oksbjerg et al., 2004; Reinecke, 2006; Wood et al., 2005). The GH-IGF system is the main endocrine growth regulator (Reinecke, 2010) (Fig 1.8) and is strongly influenced by nutritional status and environmental factors, especially temperature and light (Beckman, 2011; Deane and Woo, 2009; Duan, 1998; MacKenzie et al., 1998; Pérez-Sánchez et al., 2002; Pérez-Sánchez et al., 1995; Pickering et al., 1991; Reindl and Sheridan, 2012; Reinecke, 2010). The influence of the GH-IGF system on growth regulation is further determined by the feed intake, diet composition and stress.
GH1 is well-documented as the most potent growth stimulant in vertebrates and reviewed by Björnsson et al. (2002). This is a multifunctional hormone that also plays significant roles in other essential functions like energy and protein metabolism (Kling et al., 2012;
Mommsen, 2001; Sheridan, 1986), reproduction (Björnsson, 1997), appetite (Johnsson and Björnsson, 1994), behaviour (Björnsson, 1997; Jonsson and Bjornsson, 2002), osmoregulation, smoltification (Pelis and McCormick, 2001; Sakamoto and McCormick, 2006) and immune system (Chang and Wong, 2009; VerburgǦVan Kemenade et al., 2009).
GH1 production takes place in somatotroph cells in the anterior pituitary (adenohypophysis) and is under a strong and complex hypothalamic control via nerve cells (Fig 1.8) (Ágústsson et al., 2000; Gahete et al., 2009; Gorbman, 1995). It is called the hypothalamic-pituitary- interrenal axis (HPI-axis) and is distinct from the mammalian organisation which uses the circulatory system as a signalling link between the two brain tissues (Gorbman, 1995).
Figure 1.8. Regulation of appetite and growth in fish by ghrelin and the GH-IGF system. GH1 is produced in somatotroph cells in PIT (pituitary). GH1 release from PIT is stimulated (+) by GH releasing hormone (GHRH) and inhibited (-) by somatostatin from the hypothalamus (HYP). Circulating GH1 binds to binding proteins (GHBP) which control their availability to the receptor (GHR1) in mainly liver and muscle. Production of IGF-1 is mainly in liver and muscle. IGF-1 released to circulation also binds to binding proteins (IGFBP) before binding to receptor (IGFR1) in target organs. Binding to receptors mediates biological functions of ghrelin, GH1 and IGF-1. Main roles of ghrelin, GH1 and IGF-1 are also given. Circulating ghrelin, mainly from the stomach and gastrointestinal tract (GIT), binds to the GH secretagogue receptor (GHSR, called the ghrelin receptor) in PIT and HYP to stimulate GH1 production. The availability of GH1 and IGF-1 to their receptors is controlled by binding to circulating binding proteins (BP). GH1 also has a possible stimulating role in extrahepatic IGF-1 production. Factors analysed in thesis are enclosed with dashed lines. Modified from sources: Chang and Wong (2009); Kaiya et al. (2008); Reinecke (2010); Unniappan and Peter (2005).
Stimulatory or inhibitory signals from the hypothalamus are influenced by endogenous cues, such as nutritional state and humoral factors, and exogenous cues, such as temperature and photoperiod (Björnsson et al., 2002; Canosa et al., 2007). The hypothalamus produces the main GH1 stimulator; the GH-releasing hormone (GHRH, also called GHR factor) (Fig 1.8) and other stimulating peptides including the pituitary adenylate cyclase-activating peptide (PACAP), corticotropin-releasing hormone (CRH), neuropeptide Y (NPY), thyrotropin releasing hormone (TRH) and gonadotropin releasing hormone (GnRH) (Gahete et al., 2009).
The identification of a peripheral GH1 stimulant; ghrelin (Inui et al., 2004; Kaiya et al., 2003a; Kojima et al., 1999; Ueno et al., 2005), gave a new perspective to the GH signalling pathway and the established hypothalamic regulation (Fig 1.8). There are also indications that leptin can act as a GH secretagogue in mammals (Giusti et al., 2002) and recently in fish (Won et al., 2013), suggesting further endocrine factors possibly interacting with the GH-IGF system that should be elucidated to increase the understanding of fish growth regulation.
The main inhibition of pituitary GH1 release is caused by the somatotropin release- inhibiting factor (SRIF, or somatostatin) which is also of hypothalamic origin (Fig 1.8) (Gahete et al., 2009; Reinecke, 2010). Other inhibiting factors include the neuropeptide serotonin and the neurotransmitter norepinephrine (NE) (Gahete et al., 2009). Plasma IGF-1 also plays an important inhibiting role in regulating GH1 secretion under catabolic conditions through a negative feedback function (Duan, 1998; Reinecke, 2010). Besides,
GH1 itself is reported to have a direct negative feedback function on GH1 pituitary release in rainbow trout (Ágústsson and Björnsson, 2000). The diverse role of GH1 is reflected in the many factors involved in GH-IGF regulation and wide expression of GH1 in extra- pituitary tissue and receptors in extra-hepatic tissues (Canosa et al., 2007), which also supports a paracrine/ autocrine function of GH1 (Waters et al., 1999).
Under anabolic conditions GH1 is released into the circulatory system and can stimulate tissue growth either directly or indirectly by binding to GH receptors (GHR1 and GHR2) and stimulate synthesis and secretion of IGF-1 mainly from the liver, but also from a wide range of other tissues (Beckman, 2011; Pérez-Sánchez, 2000; Pérez-Sánchez et al., 2002;
Reinecke, 2010). The relative growth stimulating potency of IGF-1 originating from either liver or muscle is still not fully described in fish, although both promote muscle growth (Fuentes et al., 2013).
Two types of GHR exist; GHR1 and GHR2, both of which are expressed in various body tissues and fish species (Saera-Vila et al., 2007). The functions of GHR’s are still not fully understood (Saera-Vila et al., 2007), but reported results indicate that function and expression seem to be dependent on type of tissue and fish species and that there are some similarities between the GHRs (Hevrøy et al., 2015; Reindl and Sheridan, 2012). In Atlantic salmon few functional differences were found between the two GHRs, and mRNA levels in both liver and muscle tissue reacted similarly to temperature treatments of 13°C and 19°C (Hevrøy et al., 2015). In trout, liver ghr2 mRNA levels were significantly lower at 19°C compared to 13°C, while liver ghr1 mRNA was unaffected by temperature treatments (Hevrøy et al., 2015).
The availability of GH1 and IGF-1 to their receptors is controlled by binding to circulating binding proteins (BP); called GHBP and IGFBP, respectively (Fig 1.8) (Baumann et al., 1988; Clemmons et al., 1998; Duan and Xu, 2005; Kopchick and Andry, 2000; Mannor et al., 1991; Wood et al., 2005). Hence, the BPs act as a reservoir and can create a disagreement between secretion and circulating levels of GH1 as shown recently in Atlantic salmon (Einarsdottir et al., 2014). Variations in GHR1 and levels of circulating BPs can also
create an imbalance between GH1, IGF-1 and growth (Duan, 1998; Wood et al., 2005). The role of BPs in the GH-IGF system is less known and the majority of our understanding of their functions and regulation is described for mammals. In fish, BPs role is in the early phase of research and their influence on the GH-IGF actions is still poorly understood, especially for GHBP (Reindl and Sheridan, 2012). However, in recent studies of zebra fish (Kajimura et al., 2005; Kamei et al., 2008), it was found that two genes of igfbp1 (a and b) influence growth under hypoxia by binding up free IGF-1.
IGFs promote growth by stimulating growth of muscle and skeleton (bones and cartilage) and preventing breakdown of protein and cells (Duan et al., 2010; Oksbjerg et al., 2004;
Wood et al., 2005). IGF-1 is also reported to have a stimulatory effect in development, osmoregulation and reproduction (Duan, 1997; Reinecke et al., 2005).
As the name IGF-1 indicates, this endocrine growth stimulant has similar functions and structures to insulin (Duan et al., 2010). However, in terms of food availability the IGF-1 responds more to long-term changes than insulin, which reacts to short and abrupt changes (Gabillard et al., 2006; Shimizu et al., 2009). The GH-IGF system consists of both IGF-1 and IGF-2. This thesis focussed on IGF-1 (gene expressions in liver and muscle, and plasma concentration) (Fig 1.8) as it is well known to be the principle stimulant of muscle growth in fish, and the endocrine role and biological actions of IGF-2 are less understood (Reinecke, 2006). Other factors of the GH-IGF system investigated in this thesis were gene expression analyses of the receptors; GHR1 and IGFR1 in liver and muscle tissue (Fig 1.8).
Growth and IGF-1 often respond similarly to external factors, thus IGF-1 is also considered a useful growth indicator in fish in addition to GH1 (Beckman, 2011; Beckman et al., 2004;
De-Santis and Jerry, 2007; Dyer et al., 2004; Pérez-Sánchez and Le Bail, 1999; Picha et al., 2008; Wilkinson et al., 2006). Like GH1 (Chang and Wong, 2009), IGF-1 has an immune related function in fish (Segner et al., 2006; Yada, 2007; Yada, 2009), but its exact role is not yet well defined (Franz et al., 2016).
The GH-IGF regulation in fish is responsive to stressors like high temperature, fasting, handling, overcrowding and salinity (Pickering, 1993; Pickering et al., 1991; Reinecke, 2010). Generally growth reduction in relation to environmental stressors are caused by elevation of secreted stress hormones (catecholamines and corticosteroids) (Pickering et al., 1991). Reduced growth in gilthead seabream following confinement stress is thought to be caused by modifications in secretion and availability of circulating GH-IGF hormones (Rotllant et al., 2001). Also salmonids respond to handling stress by changes in circulating levels of hormones related to the GH-IGF system (Wilkinson et al., 2006), but discrepancies in results from various studies can be complicated due to different environmental conditions and sampling time regimes. When elevated levels of GH1 do not correlate with increased growth under warm conditions (Handeland et al., 2000), this may indicate an impaired growth regulation due to thermal stress (Deane and Woo, 2009; Pickering, 1993). However, due to GH1’s multifunctional role, several factors need to be considered when attempting to identify GH1 responses in growth regulation.
Atlantic salmon exposed to sudden stress episodes resulted in elevated levels of both plasma GH1 and IGF-1 along with reduced growth (McCormick et al., 1998). However, elevated plasma GH1 is also found in crowded and uncrowded rainbow trout exposed to low oxygen conditions, while crowding itself resulted in reduced plasma GH1 levels (Pickering et al., 1991).
Nutritional status and temperature can interact to stimulate the GH-IGF signalling pathway in different ways. Rainbow trout exposed to high temperature (16°C) had higher circulating GH1 levels than those at 8°C and 12°C irrespective of feeding regimes (Gabillard et al., 2003c), while plasma IGF-1 was elevated by a combination of high temperature and higher feed ration (Gabillard et al., 2003b). Postprandial peaks of both GH1 and IGF-1 are reported for various fish species (Ayson et al., 2007; Fox et al., 2009; Shimizu et al., 2009; Valente et al., 2012), and varies with factors like species, fish size, nutritional status, temperature (Reinecke, 2010). Starvation or restricted feeding generally results in increased plasma GH1, and reduced IGF-1 concentrations and hepatic mRNA levels of ghr1 and igf1, while refeeding or high feed ration reduce plasma GH1 and elevates plasma IGF-1 and hepatic
ghr1 and igf1 mRNA levels (Reinecke, 2010). Contradicting responses indicate that there may be species specific differences in how the GH-IGF system responds to fasting and refeeding or nutritional status (Reindl and Sheridan, 2012).
GHR1 declines in fasted fish, implying a reduced stimulation of IGF-1 synthesis and growth (Gray et al., 1992; Pérez Sánchez et al., 1994), but also lack of negative feedback on GH1 synthesis (Duan, 1998; Pérez Sánchez et al., 1994) leading to GH1 accumulation (Pérez- Sánchez, 2000). GH1 also has a role in metabolic use of energy from fat, which can be influenced by nutritional status and dietary energy level (Company et al., 1999; Deane and Woo, 2009; Pérez-Sánchez, 2000). Feeding a high fat diet increases the GH1 level in several fish species (Cameron et al., 2002; Company et al., 1999; Pérez-Sánchez, 2000) possibly caused by a shift in use of dietary fat as metabolic energy source rather than protein (a protein sparing effect) (Company et al., 1999; Pérez-Sánchez, 2000) or by an enhanced lipolytic activity of GH1 (Björnsson, 1997; Hevrøy et al., 2013).
The direct and interrelated actions of stimulating and inhibiting factors of the GH-IGF system are essential to describe how growth mechanisms are regulated. The tight relationship between appetite and growth regulation underlines the importance of studying both ghrelin and the GH-IGF system in order to elucidate more on these functions in fish under warm hypoxic conditions. Studies on endocrine regulation of appetite and growth are increasing in fish (Kang, 2011), but very few studies report on the interactions of the integrally linked mechanisms of the GH-IGF system and ghrelin in Atlantic salmon (Hevrøy et al., 2011; Hevrøy et al., 2012; Kullgren et al., 2013; Moen et al., 2010). Many useful parallels can be drawn from extensive mammalian studies, but variations in endocrine regulation of fish and vertebrates in general, have been reported for several functions, like appetite, growth and stress (Gahete et al., 2009; Jönsson, 2013; Jönsson et al., 2007; Kaiya et al., 2008; Pickering et al., 1991; Volkoff et al., 2010). This suggests significant species differences and a need for more endocrine studies on target fish species.
2. Aims of the thesis
The main goal of the present PhD work is to increase knowledge on appetite and growth regulation in seawater adapted Atlantic salmon in relation to high water temperature, low oxygen conditions and dietary energy concentration.
This topic is divided into three specific aims:
1. Determine pre- and postprandial nutrient dynamics and endocrine regulation of appetite and growth
2. Investigate the mechanisms by which low oxygen and high temperature impact appetite and growth
3. Determine how dietary energy concentration affects growth regulation under hypoxia and high temperature
3. Abstract of papers 3.1 Paper I
Atlantic salmon farmed in sea cage facilities are exposed to changing environmental conditions. While growth decline under lower water temperatures are reasonably well understood, the mechanisms behind production decline under high water temperatures are yet to be well elucidated and have been hampered by a lack of data describing pre- and postprandial patterns of endocrine fluctuation. The present research therefore aims to determine whether peak nutrient flux into the blood plasma is the most appropriate time point to investigate the endocrine regulation of growth and appetite under conditions of normal and high temperature, and to investigate the interrelationship between appetite and growth on a pre- and postprandial time scale. Two experiments are presented which examine ghrelin (GHRL) as an indicator of appetite stimulation, the GH-IGF (growth hormone-insulin-like growth factor) system to describe growth-regulating processes and free amino acids (FAA) to indicate postprandial nutrient influx and link appetite and anabolic processes. Postprandial sampling of plasma and white muscle tissue from short- term adapted postsmolt was conducted at 13°C and 19°C at 4, 8, 12, 16, 20 and 24 hours (h). The same samples were taken from long-term adapted big salmon at 12°C, -4, -2, -1, 0 h pre-prandially and 2, 3, 4 and 6 h post-prandially. While limited relationship between plasma ghrelin concentration and meal times was found for short term adapted postsmolt, clear ghrelin peaks were described for long term adapted salmon prior to the timing of anticipated meals. Possible explanations and consequences to experimental design in this area will be discussed. Postprandial FAA in plasma and white muscle from postsmolt were reduced at 19°C compared to 13°C and plasma levels peaked 8 h post-prandially. Muscle igf1 mRNA expression levels were consistently higher at 13°C than 19°C, with no clear postprandial patterns. In contrast, plasma IGF-1 concentration was relatively constant over time at 12°C and 13°C, but significantly declined from 20 h postprandially at 19°C. GH receptor (ghr1) mRNA expression in muscle was unaffected by temperature, peaking 4 h post-prandially at both temperatures. This paper describes growth and appetite-regulating
processes under conditions of normal and elevated temperature for Atlantic salmon, which is fundamental to our understanding of growth limitations inherent to high water temperature situations.
3.2 Paper II
This study examines how appetite and growth regulation of Atlantic salmon are affected by low dissolved oxygen (LO) and dietary digestible energy levels (DE: high [HE] vs. low [LE]). Long-term exposure to LO resulted in a reduced feed intake, growth, digestible protein and fat retention efficiencies and increased feed conversation ratio and plasma ghrelin concentrations (p < 0.05) compared to high dissolved oxygen (HO). Pair-feeding of rations based on the feed intake of the LO groups, but fed at HO, resulted in a 50% growth improvement in HE diet groups. This suggests that the poor growth under LO was not entirely caused by the reduced feed intake. Salmon adapted to LO by increased haemoglobin concentrations, while osmoregulation was affected by increased plasma chloride concentrations (p < 0.05). Plasma ghrelin concentration was unaffected by DE (p >
0.05). Growth regulation was affected by the HE diet, with increased liver and muscle growth hormone receptor ghr1 mRNA (p < 0.05), regardless of oxygen level. The growth depression due to low oxygen appears to be related to higher metabolic costs, while higher DE upregulates the GH-IGF system at the ghr1 level and found to be beneficial for growth, feed intake, oxyregulation and osmoregulation under hypoxia.
3.3 Paper III
High temperature combined with low dissolved oxygen (DO) is one of the most challenging environmental conditions farmed fish experience, thus understanding their impact on growth regulation is of relevance to cultured and wild populations. This study examines appetite and growth regulating mechanisms in Atlantic salmon postsmolt exposed to either high (HO) or low oxygen (LO) at a suboptimally high temperature (17°C). Additionally, the
effects of high (HE) and low (LE) dietary energy (DE) were examined. After a month of treatment, analyses of hormones regulating appetite (ghrelin) and growth (growth hormone receptor ghr1 and insulin-like growth factor IGF-1), and free amino acids (FAA) were measured pre- and postprandially at -4, -2, 0, 2, 4 and 6 hours (h). No pre-prandial ghrelin peaks were detected despite a significant reduction of feed intake and growth under hypoxia compared to normoxia. LO treatment also had an overall negative effect on survival compared to HO, while nutrient retention, FCR and plasma FAA concentrations were unaffected (p > 0.05). Feeding HE diet resulted in increased growth (+17%) and improved FCR (-14%) and energy retention efficiency (+26%) independent of DO. Plasma FAA concentrations were unaffected by LO treatment and DE (p > 0.05). Growth regulatory gene expressions possibly reflect an overall lower growth at a high temperature overriding the impacts of DO and DE. This study also indicates that optimal adaptation time to environmental conditions and feeding regime is crucial for establishing a regular hormonal appetite signalling that reflects real feeding anticipation in salmon.
4. General discussion
The present thesis investigates appetite and growth regulation in seawater adapted Atlantic salmon in relation to high water temperature, variable low oxygen conditions and dietary energy. This chapter compares and discusses the main findings from four fish trials which are published in three scientific papers: I (Vikeså et al., 2015), II (Vikeså et al., 2017) and III (Vikeså et al., 2016). Further implications for salmon farming and forecasted environmental challenges are also discussed.
To determine pre- and postprandial nutrient dynamics and endocrine regulation of appetite and growth, two fish trials were conducting (Paper I). In the first trial, postsmolt (196 g) were exposed to an optimal and a sub-optimally high temperature (13°C and 19°C) for 35 days (short-term adaptation) before a 24 h postprandial sampling of blood, and liver and muscle tissue were carried out. This was followed up by a long term adaptation (four months) of large salmon (3.7 kg) to feeding regimes at 12°C. The sampling set-up for large salmon was changed to separate pre- and postprandial sampling groups, to ensure postprandial sampled fish were not disturbed by the preprandial sampling, and therefore had normal feeding behaviour. Sampling range was also changed to be closer around mealtime;
from -4 h pre-prandially to 6 h post-prandially. This was to determine whether any plasma FAA or endocrine responses were rather peaking closer to mealtime, than detected by the 4- 24 h postprandial sampling regime. Pre- and postprandial analyses (-4 h to 6 h) were also conducted with short-term adapted postsmolt at a high temperature (17°C, Paper III).
To investigate the mechanisms by which low dissolved oxygen (LO, hypoxia) and high temperature regulate appetite and growth, different LO regimes were tested against groups at stable high oxygen conditions (HO, normoxia) in two fish trials (Paper II and III). In the first LO trial (Paper II), large salmon (1.3 kg) were held under a fluctuating LO regime, to mimic a possible natural situation, for 4 months at an optimal temperature for growth (12°C). A pair feeding technique was applied in order to separate the influences of oxygen
from feed intake. The second trial was conducted at a high temperature (17°C) (Paper III) with postsmolt (264 g) under stable LO and HO regimes, for 30 days.
Finally, to determine how dietary energy concentration (DE) affects growth regulation under hypoxia and high temperature, diets with high and low energy (HE and LE) were tested in both LO studies (Paper II and III).
4.1 High temperature
An elevation in water temperature has a well-known stimulatory effect on both appetite and growth within an optimal temperature range. Temperature is the main driver of metabolic rate (Brett, 1979; Jobling, 1997) and, together with feed availability, it greatly defines the potential for growth in fish. Feed intake is also important for growth as it initiates a cascade of metabolic reactions including the digestion process and secretion of regulatory factors.
However, less is known about the mechanisms behind feed intake depression and growth regulation following a further thermal elevation beyond optimum, which has been the focus of this thesis.
The signalling pathways of appetite and growth regulating hormones fluctuate and interact in response to important drivers of growth, such as feed availability and temperature.
Despite this, these dynamics have not been systematically studied in Atlantic salmon. The majority of studies investigating endocrine growth regulation at high temperature, have sampled fish at an undefined period or a single, fixed sampling point after a feeding. This approach assumes that either the endocrine or nutrient factors under investigation are similar among the experimental groups over time, or that the time point chosen yields the most informative data. In order to test these assumptions and better understand the interaction of feed intake, temperature and regulation of appetite and growth, a study on postprandial dynamics was first conducted. Fish trials at optimal and high temperatures (Paper I and III) with up to 24 h postprandial sampling regimes were therefore designed to detect peak times for circulating FAA and endocrine growth factor levels in relation to meal time. The aim was to define the most appropriate time point to investigate regulation of growth and appetite and to get a basic understanding of how these mechanisms are regulated and interact in relation to temperature in Atlantic salmon (Paper I).
